Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition

Abstract

Analysis of peptides by reverse-phase highpressure liquid chromatography would be simplified if retention times could be predicted by summing the contribution to retention of each of the peptide's amino acid side chains. This paper describes the derivation of values ("retention coefficients") that represent the contribution to retention of each of the common amino acids and end groups. Peptide retention times were determined on a Bio-Rad "ODS" column at room temperature with a linear gradient from 0.1 M NaCIO4, pH 7.4 or 2.1, at 0 min to 60% acetonitrile/0.1 M NaCIO4 at 80 min. The NaClO4, a chaotropic agent, was added to improve peak shape and to minimize conformational effects. Retention coefficients for the amino acids were computed by using a Hewlett-Packard 9815A calculator programmed to change the retention coefficients for all amino acids sequentially to obtain a maximum correlation between actual and predicted retention times. Correlations of 0.999 at pH 7.4 and 0.997 at pH 2.1 were obtained for 25 peptides including glucagon, oxytocin, [Metlenkephalin, neurotensin, and somatostatin. This high degree of correlation suggests that, for peptides containing up to 20 residues, retention is primarily due to partition processes that involve all the residues. Although steric or conformational factors do have some effect on retention, the data suggest that under the above chromatographic conditions the retention of peptides containing up to 20 residues can be predicted solely on the basis of their amino acid composition. This possibility was tested by using data taken from the literature.The possibilities for separating and isolating small peptides have been markedly improved by the introduction of reverse-phase high-pressure liquid chromatography (HPLC) (1-5). This technique depends upon the hydrophobic interactions between a hydrocarbonaceous column and the peptides to be separated: the more hydrophobic (lipophilic) the compound, the stronger its retention by the column. To elute strongly retained compounds, aqueous solutions (the "mobile phase") containing a large amount of organic solvent must be pumped through the column. Choice of the optimum mobile phase and chromatographic conditions for given peptides can be found by trial and error after qualitatively examining the balance of hydrophobic and hydrophilic amino acids present in the peptide. However, as noted by Molna'r and Horvath (1), it should be possible to obtain quantitative estimates of the hydrophobicity of the amino acids contained in a peptide, which will reflect their retention on the reverse-phase column. Estimates of hydrophobicity based on octanol/water partition coefficients exist for many but not all amino acids. By using such values, O'Hare and Nice (2) noted that the retention order for small peptides was generally correlated with the sum of the values for the most hydrophobic residues of the peptides. The many deviations they found between the observed order of elution and the lipophilicity estimates presumably derive from th...

Aspartate‐Bond Isomerization Affects the Major Conformations of Synthetic Peptides

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